Engineered heterodimeric proteins
Inventors
CEBE, Regis • IRIGARAY, Sebastien • SKEGRO, DARKO
Assignees
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Publication Number
US-11685776-B2
Publication Date
2023-06-27
Expiration Date
Abstract
The present invention provides heterodimeric antibodies and fragments thereof and methods for their preparation, wherein the pairing of heavy and light chains has been improved. Interface residues were mutated such that each light chain strongly favoured its cognate heavy chain when two different heavy chains and two different light chains were co-transfected and co-expressed in the same cell to assemble a functional, heterodimeric antibody or fragment thereof.
Core Innovation
The invention addresses mispairing of heavy and light chains in co-transfected/co-expressed heterodimeric antibodies, including bispecific IgG formats. It focuses on controlling heavy chain variable domain (VH) and constant domain 1 (CH1) pairing with the corresponding light chain variable domain (VL) and constant domain (CL) to favor correct heterodimerization and reduce incorrect pairing.
Electrostatic steering is achieved by mutating interface residues at EU-numbered positions in the VH/CH1 and VL/CL. The engineered charged residues in the VH/CH1 and VL/CL pairwise form an interface that is electrostatically favourable to heterodimerization, described as an electrostatic “clip-effect” that stabilizes the CH1–CL interaction.
The description further includes engineered residue sets for different light-chain types, including kappa vs lambda, and combines charge engineering with CH1–CL interface disulfide-bond engineering. In particular, a VH–VL interchain disulfide is engineered to covalently lock correct HC/LC pairing, and mispairing detection is simplified using electrophoresis.
Claims Coverage
The independent claim provides a charged-interface framework with an electrostatically favourable VH/CH1–VL/CL heterodimerization interface based on EU-numbered acidic/basic residues, supported by dependent claim features including specific engineered residue sets, quantitative heterodimerization performance thresholds, additional Fab extensions with different charge assignments, and an engineered VH–VL interchain disulfide replacement.
Electrostatic interface pairing in VH/CH1 and VL/CL for heterodimerization
A heterodimeric IgG antibody or fragment comprising at least two Fabs, wherein engineered VH and CH1 comprise an acidic amino acid at EU position 39, a basic amino acid at EU position 147, and an acidic amino acid at EU position 165, and engineered VL and CL comprise a basic amino acid at EU position 38, an acidic amino acid at EU position 124, and a basic amino acid at EU position 169 or 170, or wherein the roles of acidic/basic residues are interchanged; wherein the amino acids in the VH/CH1 and VL/CL domains pairwise are of opposing charge and correspond to form an interface electrostatically favourable to heterodimerization, wherein the acid amino acid is aspartic acid (D) or glutamic acid (E), the basic amino acid is arginine (R), lysine (K) or histidine (H), and the numbers are according to EU numbering.
Heterodimerization threshold of at least 95% measured by LC-MS
A heterodimeric IgG antibody or fragment that achieves at least 95% heterodimerization as measured by liquid chromatography–mass spectrometry.
Heterodimerization up to 100% measured by LC-MS
A heterodimeric IgG antibody or fragment that achieves heterodimerization of up to 100%, as determined by liquid chromatography–mass spectrometry.
Additional Fab with different charged interface assignments for electrostatically favourable pairing
A heterodimeric IgG antibody or fragment comprising an additional Fab with engineered VH/CH1 and VL/CL domains containing acidic and basic amino acids at EU-numbered positions so that opposing charges at the pairing interface electrostatically favour heterodimerization, wherein the charge assignments at EU positions 39, 147 and 165 are different between the engineered VH of one Fab and the engineered VH of the additional Fab, using EU numbering.
Engineered VH–VL interchain disulfide replacing an interchain disulfide in an additional Fab
A heterodimeric IgG antibody or fragment wherein, in an additional Fab, an interchain disulfide bond is replaced by an engineered VH–VL interchain disulfide bond.
Overall, the claims cover heterodimeric IgG antibody formats whose VH/CH1–VL/CL pairing is driven by opposing charged residues at EU-numbered interface positions to form an electrostatically favourable heterodimerization interface. Dependent inventive features further specify quantitative LC-MS heterodimerization expectations, extension to an additional Fab with different charge assignments, and covalent locking by replacing an interchain disulfide with an engineered VH–VL interchain disulfide.
Stated Advantages
Improved correct heavy/light chain pairing for heterodimeric Fab/IgG constructs, reported as reaching 95–100% correct pairing for engineered mutation sets.
Stabilization of the CH1–CL interaction via an electrostatic “clip-effect” that favors heterodimerization.
Simplified mispairing detection using electrophoresis.
Documented Applications
Engineered heterodimeric antibodies including bispecific IgG formats produced by co-transfection/co-expression, addressing mispairing of heavy and light chains.
Multispecific Fab arms with engineered charged Fab arms having differing charge assignments.
Binding and thermal stability characterization of engineered antibody constructs using BIAcore and DSC.
Detection and quantification of correct HC–LC pairing and mispairing using LC-UV-ESI-MS and electrophoresis.
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